The Unit of Molecular Signal Transduction investigates signal transduction pathways that mediate the actions of hormones and growth factors in mammalian cells, with special emphasis on the role of phosphoinositide-derived messengers. Phosphoinositides are a small fraction of the cellular phospholipids, but play a critical role in the regulation of many (if not all) signaling protein complexes that assemble on the surface of cellular membranes. Phosphoinositides regulate protein kinases and GTP-binding proteins, as well as membrane transporters including ion channels, thereby controlling many cellular processes such as proliferation, apoptosis and metabolism. Our group focuses on one family of enzymes, the phosphatidylinositol 4-kinases (PI4Ks) that catalyze the first committed step in phosphoinositide synthesis. Current studies are aimed at (1) understanding the function and regulation of several phosphatidylinositol (PI) 4-kinases in the control of the synthesis of hormone-sensitive phosphoinositide pools; (2) characterizing the structural features that determine the catalytic specificity and inhibitor sensitivity of PI 3- and PI 4-kinases; (3) defining the molecular basis of protein-phosphoinositide interactions via the pleckstrin homology and other domains of selected regulatory proteins; (4) developing tools to analyze inositol lipid dynamics in live cells; (5) determining the importance of the lipid-protein interactions in the activation of cellular responses by G protein-coupled receptors and receptor tyrosine kinases. One of the most important regulators of phosphoinositide levels are the phospholipase C (PLC) enzymes that hydrolyze the lipid, phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]. Of the various isoforms of PLCs, the delta forms are the most evolutionarily conserved. These enzymes are believed to be recruited to membranes and regulated by their lipid substrate, PI(4,5)P2, via interaction with their pleckstrin homology (PH) domain, a protein module that is characterized by high-affinity binding to phosphoinositides. Recent studies have identified a new isoform of PLCdelta, the delta4 enzyme, and indicated that its PH domain selectively binds the lipid PI(4,5)P2, but not the soluble inositol 1,4,5-trisphosphate (IP3). To understand the role of the PLCdelta4 PH domain in the localization and function of PLCdelta4 enzyme, the inositol-lipid and inositol phosphate binding properties of the full-length enzyme, and its isolated PH domain, were analyzed in comparison with the similar features of the PLCdelta1 protein. Our studies showed that the PH domain of PLCdelta4 indeed has a lower affinity to both the lipid, PI(4,5)P2 and the soluble IP3, but still could recognize PI(4,5)P2 in the plasma membrane when expressed as a GFP fusion protein in live cells. In contrast, the full-length PLCdelta4 was found not to be associated with the plasma membrane but primarily with the endoplasmic reticulum, and removal of the PH domain from the enzyme failed to affect its cellular localization. These data suggest that the PH domain of PLCdelta4 is not responsible for the localization of the enzyme, probably because it is not fully exposed in the molecule. Our data suggest that in spite of their similar architecture, the PLCdelta1 and -delta4 enzymes are differentially regulated by their PH domains. As a result of PLC activation, IP3 is generated from membrane PI(4,5)P2 upon stimulation of specific forms of cell-surface receptors. IP3 rapidly binds to its intracellular receptors and releases Ca2+ from non-mitochondrial Ca2+ stores to produce the cytoplasmic Ca2+ increase that triggers specific cellular responses of the target cell. IP3Rs are functional Ca2+ channels that work as homo- or heterotetramers. Each receptor subunit has a channel portion containing six transmembrane helices and a pore-domain located between TM5 and TM6, close to the C-terminus of the protein. The ligand-binding domain (LBD) of the receptor is located near the N-terminus of the molecule, and is separated from the channel domain by a long intervening regulatory region facing the cytoplasm. In spite of intense investigations, little is known about the manner in which the binding of IP3 to the N-terminal LBD affects the channel gating properties of the molecule. To address this issue, we investigated whether the LBD of the InsP3R acts as a tethered adaptor module that regulates the channel activity via InsP3-induced conformational changes. For this, we used a novel molecular approach in which the isolated LBD of the type-I InsP3 receptor or its components were tethered to the cytoplasmic surface of the endoplasmic reticulum (ER), where IP3Rs reside, and the effects of their expression on Ca2+ signaling were compared to those of the same constructs expressed in the cytoplasm. When the IP3R-LBD was expressed with a C-terminal short hydrophobic sequence that targets it to the cytoplasmic surface of the ER, it strongly inhibited agonist-induced Ca2+ signaling. Interestingly, the inhibitory effect of the ER-tethered IP3R-LBD form was not due to IP3 binding but rather was the result of the emptying of the IP3-sensitive Ca2+ stores. Another ER-tethered structurally unrelated InsP3 binding module, the PH domain of the PLC-like p130 protein, did not show such an effect. These data suggested that the IP3R-LBD has structural elements that are capable of releasing Ca2+ from the ER. To determine whether the release of Ca2+ occurs via the IP3Rs, we used DT40 cells that lack all three isoforms of the receptor. Expression of the ER-tethered IP3R-LBD had no effect on the intracellular Ca2+ stores in the triple-knockout cells, while it depleted the stores in the wild-type DT40 cells, indicating that the LBD interacts with the endogenous IP3Rs and leads to their opening. In further experiments we determined that the all-helical domain of the LBD was sufficient to open the channel even though it alone fails to bind IP3, and that the addition of an inhibitory N-terminal segment to the LBD (residues 1-224) greatly decreased its efficacy to release Ca2+. Based on these data, we propose that the LBD of IP3Rs is positioned in close proximity to the channel domain, and that exposure of the all-helical domain from a cryptic state is part of the mechamism by which IP3 binding leads to channel activation. These data provide an important clue to the regulation of IP3R channel function, and open new directions to clarify the molecular details of this process.